Vol. 11: 289–294, 2011 AQUATIC BIOLOGY Published online February 23 doi: 10.3354/ab00310 Aquat Biol

Seasonal variation in the occurrence of the medicinal leech Hirudo orientalis in Guilan Province, Iran

K. Darabi-Darestani, M. Malek*

University of Tehran, School of Biology, College of Science, University of Tehran, Tehran, Iran

ABSTRACT: Two small populations of medicinal leeches Hirudo orientalis Utevsky & Trontelj, 2005 were sampled monthly over the course of a year (November 2008 to October 2009) in 2 separate regions of Guilan Province in northern Iran. Environmental factors, including host availability, tem- perature and vegetation density (biomass in g km–2) were analyzed to assess their impact on leech populations. The study areas supported only low densities (ind. km–2) of medicinal leeches, mostly due to agricultural activity which has caused habitat destruction and has gradually limited the distri- bution of leeches to small, patchy bodies of water. Agricultural activity is seasonal, so leeches are not affected by this activity equally year round. Leeches were most abundant in May/June, were present in small numbers in July/August, and hibernated from December to March 2009 at both sites. Leech density was significantly correlated with amphibian density (biomass in g km–2), the major hosts in the sample areas. Temperature and aquatic vegetation densities affected leech numbers directly (temperature influencing leech growth rate and vegetation providing shelter from potential preda- tors) and indirectly (due to the impact of temperature and vegetation on the amphibian population, the leeches’ major prey). Leeches of <1 g were found mostly in April and May following hatching, while those >5 g were predominant in September.

KEY WORDS: Seasonal variation · Environmental factors · Population density · Hirudo orientalis · Iran

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INTRODUCTION and the ecological distribution of Hirudinea, the popula- tion dynamics of H. hainana, the adaptation of H. hainana Throughout nearly 2000 yr of documented history, the to temperature, soil humidity and pH, the response of H. medicinal leech Hirudo medicinalis L. has received con- hainana to temperature, relative humidity and light, and siderable attention in various fields of human activity. Its the effects of human activities on H. hainana populations ectoparasitic, haematophagous feeding habit has been have been discussed (Enguang 2008). H. orientalis used and misused for medicinal purposes for centuries Utevsky & Trontelj, 2005, another medicinal leech, is (Utevsky & Trontelj 2005). H. medicinalis was consid- similar in appearance to H. medicinalis L. Recent mole- ered endangered by the Convention on International cular systematic research (Trontelj et al. 2004, Trontelj Trade in Endangered Species of Wild Fauna and Flora & Utevsky 2005) confirmed the validity of the taxonomic (CITES) in 1987 (see Kasparek et al. 2000). Utevsky & classification of 2 previously neglected species, H. ver- Trontelj (2005) regarded H. medicinalis as the best-stud- bana Carena, 1820 and H. troctina (Johnson, 1816), and ied leech species, but only limited work has been carried suggested that a different variety from Transcaucasia, out to determine seasonal variation in its occurrence Iran and Uzbekistan is a new species (Utevsky & Trontelj (Wilkin & Scofield, 1991, Demirsoy et al. 2001, Kalninˇ ¸ s 2005). Grosser & Peˇsi´c (2006) formally described the 2006). The growth of the medicinal leech H. medicinalis medicinal leech H. orientalis found in Mazandaran L. was studied in the wild using a mark and recapture Province, Noshahr, Iran. There is little information avail- technique and in the laboratory (Wilkin & Scofield 1991). able on H. orientalis, but, as it is in high demand for med- Hirudinea in China were reviewed in Enguang (2008), icinal purposes and collected from this and other regions

*Corresponding author. Email: [email protected] © Inter-Research 2011 · www.int-res.com 290 Aquat Biol 11: 289–294, 2011

in large numbers (K. Darabi-Darestani pers. obs.), it may study areas are near rice fields, and temporary agri- also be endangered. Although there is no published data cultural activities might affect leech habitats during to support this assumption, leech farming would be a the year, for example, when farmers construct irriga- good solution to avoid over-exploitation of leeches tion channels that remain open for a month. The San- should they indeed be endangered. Currently, there is gar site did not experience much fluctuation because no documentation on the seasonal distribution of H. ori- it was suitably located such that water could pass entalis. The aim of the present study was to examine 2 through it during the month of agricultural activity small populations of H. orientalis and to determine the and there was no need for the farmers to disturb it. effects of environmental factors on seasonal fluctuation The Rasht site, however, was disturbed since chan- in life stage and numbers. nels were dug during the agricultural month to let water flow through. Sampling and laboratory methods. Both areas were MATERIALS AND METHODS surveyed monthly from November 2008 to October 2009 using a semi-quantitative method (Elliott & Tul- Study area. To study seasonal variation in the oc- lett 1986). Surveys were made in all months; however, curence of Hirudo orientalis, 2 populations were se- no specimens were found in the cold months. Leeches lected in Guilan Province, northern Iran (Fig. 1). The were attracted by disturbing the water and then hand- first sample site near the city of Rasht (37° 16’ N, collected for 1 h to give a simple value for abundance. 49° 36’ E), consisted of 4 areas of shallow still waters The ‘collecting efficiency’ was calculated as the num- (2.0 to 4.5 m depth) with a total surface area of 2 × ber of leeches collected per hour by a single collector 2.8 km2. These patchy bodies of water are near rice (Demirsoy et al. 2001). Leeches were weighed individ- fields and support freshwater life throughout the ually to the nearest 0.01 g in the laboratory and year. Their water supply comes from annual precipi- grouped into 3 classes (<1, 1 to 5, and >5 g), represent- tation (mean 1359 mm per annum). The second site in ing hatchlings, juveniles and adults, respectively. No Sangar, a small town (37° 27’ N, 48° 35’ E) near Rasht, fed leeches were present in this study because they was a closed, shallow (2.4 m depth) water passage showed no interest in the collector as a potential prey. <1 × 2.5 km. During agricultural activity, a narrow There was usually a trace of blood in medicinal shallow stream of slow-flowing water passes through leeches’ digestive tract; however, it was not enough to it for one month (July) a year. Similar to the Rasht affect the wet weight measurement and determination site, this site also had access to a regular supply of of the life cycle stage of the individuals studied. freshwater from rainfall and shallow muddy water The major hosts for leeches in both areas were throughout the year. Other areas in Guilan Province amphibians. Amphibians and surface vegetation were were also investigated but no leeches were found. collected by net for 1 h, and the biomass calculated. All The 2 study areas were separated by 18 km. The data relating to biomass of surface vegetation, leeches and amphibian are relative figures obtained for 1 h each month and have been compared between differ- ent months and stations for the purposes of this study. Dominant amphibians in both study areas included a Bufo sp. and a Rana sp., both of which were collected at the same time as the leeches. Predation of leeches on amphibians was observed during the course of the study, especially in warm months. However, these leeches were not collected because they were fed and did not attack the collector, and thus did not meet the collecting efficiency criteria. Predominant vegetation at both sites included algae, mosses, and common floating pond plants such as duck weed (Lemnaceae), common frogbit Hydrocharis species, waterthread pondweed Potamogeton species, and water nymph Najas species. Vegetation densities were grouped into 2 wet biomass classes of high and low density. During the processing of all samples, care was taken to ensure Fig. 1. (a) Location of Guilan Province in the north of Iran that surplus water was removed before weighing. Dis- (dark grey area). (b) Guilan Province. Black dots indicate solved water oxygen and pH were measured using a study areas (from www.gilanvila.com/gilan_map.html) multifunction WTW MULTILINE P4 device. Darabi-Darestani & Malek: Seasonal variation in Hirudo orientalis 291

Statistical methods. All data were analyzed by SPSS 17.0. Kolmogorov-Smirnov normality tests were applied to test the normal distribution of the data and parametric or nonparametric tests were used as appropriate.

RESULTS

Physical characteristics of study areas

In Rasht, the substratum was muddy. Dissolved oxy- gen showed little change during the year, remaining at 7.75 to 7.98 mg l–1. Air temperatures at both sites showed little difference, < 2°C. Leeches were most abundant in May/June (at water temperatures of 15 to 23°C). The pH was almost constant throughout the year (7.2 to 7.3). In Sangar, the same chronological pat- tern of leech abundance was observed (at water tem- peratures of 14 to 21°C in May/June). The substratum was muddy with a high levels of sedimentation. Dis- solved oxygen showed little change during the year Fig. 2. Hirudo orientalis. Fluctuations in the mean total wet (6.84 to 7.26 mg l–1). The pH was almost constant at biomass of leeches during all months for both study areas. *: no specimens present 7.1 to 7.2. A t-test showed no significance difference in the mean number of leeches in the 2 areas of study (p = 0.2, t = 1.165). months in both study areas (in Rasht: χ2 = 115.4, p < 0.0005; in Sangar: χ2 = 78.4, p < 0.0005).

Seasonal variation in leech wet biomass Seasonal variation in leech weight classes Seasonal variation in the mean wet biomass in both study areas is given in Table 1. The maximum weight Rasht of a Hirudo orientalis individual (5.81 g) was obtained at Rasht in September 2009. Sharp reductions in total In Rasht no uniformity in distribution of the weight leech mean wet biomass in Rasht occurred during July classes was observed during the year (Fig. 3a). Juveniles 2009 (Fig. 2). The highest mean wet biomass (159.09 g) were significantly more frequent than the other classes was obtained at Rasht in June 2009, and the lowest (mean wet weight of leeches = 3.08 g). Hatchlings were (31.65 g) was obtained at Sangar in October 2009. most frequent in April and May 2009. In November There was a significant difference between mean 2008 and June and July 2009 juveniles were dominant. weight (g) of leeches in both study areas (Mann- Adults were most frequent in September 2009 (χ2 = 5.40). Whitney U = 12533.50, p = 0.008), and a significant dif- ference in the mean leech weight (g) in different Sangar Table 1. Hirudo orientalis. Wet weight (g) of leeches from 2 sample sites in northern Iran; no data for December to March In Sangar, juveniles were the most frequently observed group throughout the year (Fig. 3b). The 2 Month Rasht Sangar hatchlings were dominant in April and May (χ = 0.60), Mean ± SE Range Mean ± SE Range while adult leeches were most abundant in August and September 2009 (χ2 = 5.40). The frequency of juveniles Nov 2008 2.98 ± 1.07 1.73 –5.29 2.97 ± 1.25 1.59–5.14 (χ2 = 2.79) was the same as in Rasht with the exception Apr 2009 0.59 ± 0.30 0.10–0.99 0.60 ± 0.27 0.11–0.97 May 2009 2.38 ± 1.45 0.18–4.88 1.43 ± 1.50 0.11–4.71 of May 2009. Greater variation in leech weight classes Jun 2009 3.13 ± 1.10 1.01–4.93 2.61 ± 1.16 1.12–4.73 was seen in Rasht. A chi-square test and contingency Jul 2009 3.40 ± 1.03 1.33–4.71 2.62 ± 0.84 1.44–4.20 table showed no significant difference in the total Aug 2009 4.00 ± 1.39 1.83–5.81 3.63 ± 1.30 1.62–5.71 number of adults between the 2 study areas (χ2 = 2.64, Sep 2009 5.40 ± 0.27 5.02–5.81 5.19 ± 0.18 5.01–5.71 Oct 2009 3.43 ± 0.96 1.81–5.03 2.87 ± 1.42 1.09–5.16 p = 0.45), but the difference was significant in some months (χ2 = 822.54, p < 0.0005) (Fig. 3). 292 Aquat Biol 11: 289–294, 2011

Fig. 4. Correlation between amphibian and leech density

Effect of vegetation biomass on leech biomass

There was a significant correlation between leech and vegetation biomass in both study areas (r = 0.90, p < 0.0005) (Fig. 6a). In Rasht, mean wet biomass of vegetation was 238.77 g km–2, which was greater than at Sangar (χ2 = 213.19), but the difference was not sig- nificant. Fig. 6b shows that there was a close relation- ship between amphibian and vegetation biomass fluc- tuations as determining factors in leech abundance Fig. 3. Hirudo orientalis. Monthly variations in the frequency of life stages in (a) Rasht and (b) Sangar. *: no specimens present

Amphibian density and leech density

Leech number (r = 0.942, p < 0.0005) and wet bio- mass (r = 0.88, p < 0.0005) showed a close and signifi- cant correlation with amphibian density in numbers and wet biomass, respectively, and were directly af- fected by fluctuations in amphibian numbers/biomass (Figs. 4 & 5). Density of amphibians in Rasht (23.67 ind. km–2 and 620.09 g km–2, respectively) was higher than in Sangar (19.16 ind. km–2 and 577.86 g km–2, respec- tively) during all seasons; however, the differences were not significant. Amphibians were abundant throughout the year except in the cold months, includ- ing January, February and March, when both areas were covered with snow. Amphibian density showed no significant difference between localities. There was a significant correlation between the densities of Fig. 5. Correlation between amphibian and leech mean total leeches and amphibians (r = 0.94, p < 0.05) (Fig. 4). wet biomass Darabi-Darestani & Malek: Seasonal variation in Hirudo orientalis 293

density (the only available food source for leeches) and vegetation. Correlation between temperature and total leech biomass is shown in Fig.7. A significant correla- tion was found between temperature and leech wet biomass (r = 0.75, p < 0.05).

DISCUSSION AND CONCLUSIONS

To date, no data have been available on seasonal variation in Hirudo orientalis populations in Iran. Results of the present study confirmed the presence of 2 small populations of this species in the Guilan Province. A sharp reduction in total leech mean wet biomass in Rasht occurred during July 2009, when the area was disturbed due to agricultural activities. Hatchlings were predominant in April and May. Demirsoy et al. (2001) showed that the number of leeches in this weight class increases strongly during May and June and reaches a maximum in July. A con- tinuous drop was observed in Rasht after August 2009, associated with low water temperature, reduced am- phibian density, and lower vegetation biomass. H. ori- entalis were observed to hibernate during the cold months. Another condition affecting leech density is the amount of rooted vegetation relative to the volume of water (Mann 1962). Small ponds have a large pro- portion of rooted plants, and the products of their decay lead to a high concentration of humic acids in the water (Tucker 1958). However, in the present study the effect humic acid and its accumulation as a factor affecting the leech community was not measured.

Fig. 6. Correlation between mean total wet biomass of vegeta- tion and (a) Hirudo orientalis and (b) amphibians

(subsequently affecting leech biomass). Vegetation acts as a shelter for both amphibians and leeches. There was a significant correlation between amphibian and vegetation wet biomass (r = 0.95, p < 0.0005). Vegeta- tion biomass showed fluctuation during the course of the study in both sampling areas.

Effect of temperature on leech biomass

No temperature differences were observed between the 2 study areas. Air temperature was found to be a Fig. 7. Hirudo orientalis. Correlation between mean total wet dominant factor affecting the amphibian population biomass of leeches and temperature for all months 294 Aquat Biol 11: 289–294, 2011

Vegetation provides shelter and food for tadpoles, Acknowledgements. The authors thank Dr. Sari for his advice which then become prey for leeches in their juvenile and H. Salehi, M. Parsa, and A. Kazemi for their help. We also thank the Research Affairs Office of the University of Tehran and adult stages, as was observed in this study. Fur- for financial support. We are grateful to Lucidus Consultancy thermore, vegetation provides shelter for leeches, for critical comments and English editing of the earlier version which helps protect them from predators such as ducks. of the manuscript. Therefore, surface vegetation increased the chances of LITERATURE CITED leech survival both directly and indirectly. Mean wet biomass of leeches increased significantly with an in- Araoye PA (2009) The seasonal variation of pH and dissolved crease in vegetation biomass. oxygen (DO2) concentration in Asa lake Ilorin, Nigeria. 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Editorial responsibility: J. Rudi Strickler, Submitted: May 21, 2010; Accepted: December 6, 2010 Milwaukee, Wisconsin, USA Proofs received from author(s): February 10, 2011